Milk Production Management & Dairy Development

Lesson 18. CLONING, TRANSGENIC ANIMALS, EMBRYO SEXING ETC

Module 12. Biotechnology in dairy animals production

Lesson 18
CLONING, TRANSGENIC ANIMALS, EMBRYO SEXING ETC

18.1. Introduction

Biotechnology is a process applied for exploitation and control of biological system achieved with the help of microorganisms and cells taken from plants and animals through integration of several disciplines including microbiology, biochemistry, genetics and biomedical engineering. Biotechnology gives us the unprecedented opportunities to improve reproductive efficiency of dairy animals and reproduction technology in enhancing genetic gain through artificial insemination, multiple ovulation, Marker assisted selection, embryo sexing, animal cloning, pregnancy diagnosis and transgenic animals and sexing of semen.

18.2 Cloning

Cloning is a tool in the animal biotechnology toolbox, which includes artificial insemination, embryo sexing and in-vitro fertilization. A clone is a genetically identical animal that can be produced either by embryo splitting (as occurs in nature) or transfer of embryonic blastomeres or somatic cells as donor nuclei. Somatic Cell Nuclear Transfer(SCNT) is a powerful technique and potentially used for the multiplication of desired animals, genetically modified farm animals to make modification of micro growth, disease resistance etc, superior gene type could be multiplied by cloning and if required the technique can be combined with transgenic (for fertility and other desired traits) to produce desired bulls. Recently the SCNT has emerged as a tool for production of stem cells for therapeutic purpose popularly known as therapeutic cloning.

The success rate in terms of pregnancy establishment varies from 25-52% up to 19% pregnancies reach up to the term and 2-16% of embryos develop to healthy calves at weaving.

18.2.1 Applications of cloning

1. Bio-medical applications, such as the production of pharmaceuticals in the blood or milk of transgenic cattle.

2. Cloning may also be useful in the production of research models. These models may or may not include genetic modifications.

3. Uses in agriculture include many applications of the technology which include:

a) Making genetic copies of elite seed stock and prize winning show cattle.

b) Other purposes may range from "insurance" to making copies of cattle that have sentimental value, similar to cloning of pets.

c) Increased selection opportunities available with cloning may provide for improvement in genetic gain.

d) The ultimate goal of cloning has often been envisioned as a system for producing quantity and uniformity of the perfect dairy cow. However, only if heritability were 100%, would clone mates have complete uniformity. Changes in the environment may have significant impact on the productivity and longevity of the resulting clones. Changes in consumer preferences and economic input costs may all change the definition of the perfect cow. The cost of producing such animals via cloning must be economically feasible to meet the intended applications.

18.2.2 Limitations

1 Present inefficiencies limit cloning opportunities to highly valued animals. Improvements are necessary to move the applications toward commercial application.

2 Cloning has additional obstacles to conquer. Social and regulatory acceptance of cloning is paramount to its utilization in production agriculture. Regulatory acceptance will need to address the animal, its products, and its offspring.

3 Loss of pregnancies due to developmental abnormalities. These abnormalities appear to be due to faulty epigenetic reprogramming ad gene expression in the genes of different cells.

18.3 Trans Genesis

Incorporation of foreign gene into the genome of an animal led to the development of the transgenic technology and the animal, thus produced is called as transgenic animal

18.3.1 Somatic cell nuclear cloning

Cloning is a route for the production of transgenic livestock which allows for the homologous insertion of DNA sequences resulting in the opportunity to target the alteration or removal of endogenous genes. The cost of production of transgenic livestock had been high and thus limited involvement of laboratories worldwide in research in this area.

18.3.2 Embryonic stem cell mediated gene transfer

This involves prior insertion of the desired DNA sequence by homologous recombination into an invitro culture of embryonic stem cells. Stem cells are undifferentiated cells that have the potential to differentiate into any type of cell and therefore to give rise to a complete organism. These cells are then incorporated into an embryo at the blastocysts stage of development. Embryonic stem cell mediated transfer is the method of choice for gene inactivation, the so called knockout method.

18.3.3 Embryo micromanipulation and sexing

Most basic studies in maintaining embryos have been done in mice but application of these new techniques to large domestic animals will make them a powerful tool in animal breeding.

The ability to determine the sex of embryos prior to transfer to recipients has commercial application to the embryo transfer industry especially in relation to the production of females for dairy development programme. Male animals are also not less important or the superior male animals can be maintained for semen collection and or providing the natural service to the animals in village conditions especially in developing countries like India where MOET is not expected to be used in field conditions in coming years. Since, sex of an individual is set at fertilization and depends on whether the ‘X’ bearing ovum is fertilized by a ‘Y’ or ‘X’ bearing spermatozoa. Therefore sex predetermination can be achieved by separation or differential activation of ‘Y’ and ‘X’ spermatozoa using differences between spermatozoa such as mass, motility or sex has not been perfected so far.

18.3.4 Applications of transgenic animals

A wide gamut of applications is possible which includes:

• Gene Pharming: Refers to the production of recombinant biologically active human proteins in the mammary glands of transgenic animals.

• Antibody production: Numerous monoclonal antibodies are being produced in the mammary gland of transgenic goats that can be employed both for diagnosis and treatment of diseases.

• Xenotransplantation: Producing transgenic animals to meet the demand for organs and tissues that need to be transplanted.

• Blood replacement: Here the whole idea is to produce functional human hemoglobin in transgenic animals.

• Disease model: The traditional lab experimental animal mouse differs in physiology, anatomy and life span from those of humans, therefore making it an inappropriate model for many human diseases. One the other hand, farm animals, such as pigs, sheep or even cattle, may be more appropriate models to study potential therapies for human diseases which require longer observation periods.

• Carcass composition: Transgenic animals are being produced, having significant improvements in economically important traits such as growth rate, feed conversion and body fat/muscle ratio.

• Lactation: Emphasis on transgenic animals with improved physicochemical properties of the milk. Hypoallergenic milk, milk with modified composition of casein are few examples.

• Eco-friendly: Environment friendly transgenic animals like creation of transgenic animals can convert certain pollutant materials into nonpolluting products.

• Wool production: To increase the amount and quality of fleece produced by animals.

• Disease resistance: To create transgenic animals that can resist diseases better.

Application of cloning, production of desirable animals by using transgenic technology and determination of sex of the embryo will increase the products with of the animals but the costs involved in their implementation were high and it takes some more time to reach to the field level.

18.4 Embryo Sexing

Fig. 18.3(a) Embryo sexing procedure

Fig. 18.4(b) Embryo sexing Procedure

18.4.1 Steps in embryo sexing

1 Collection of embryos produced in vitro or in vivo.

2 Selection of grade one or grade two embryos.

3 Embryo washed with PBS & placed in a drop containing 200 mM sucrose under micromanipulator.

4 Zona pellucida cut open with fine micro blade.

5 Few blastomere sucked with fine aspiration pipette.

6 Washed in KCl & transferred to Eppendorf tube.

18.4.2 Isolation of embryonic DNA

1 Biopsy in 0.5 ml Eppendorf tube + Proteinase-K + 9 μL of lysis buffer.

2 Overlaid with 25 μL of mineral oil

3 Incubated at 37°C for 10- 60 min

4 Inactivation of proteinase-K at 98°C for 10 min.

5 Cooled at 4°C

18.4.3 Amplification of DNA

1 15 μL of PCR reaction mixture(PCR reaction buffer, primers, 1.5 μL of Taq DNA polymerase & 125 μg of Ethidium bromide) is added to the tube

2 Subjected to PCR cycling

3 3 min. denaturation at 94°C è 10 cycles of denaturation at 92°C è Annealing at 50°C (80 seconds) è Extension at 72°C for 20 seconds èFurther 40 cycles at 60°C of annealing temperature èFinal extension achieved by 5 min. incubation at 72°C

18.4.4 Identification of sex: It can be done by two approaches

1 Electrophoretic method-- In PCR second pair of primer added to increase accuracy After electrophoresis Y-specific bands are observed Autosomal primer commonly used is C1C2

2 Direct observation under UV light: tubes having male DNA show bright pink fluorescence.

References

1 V.D. Mudgal, KK. Singhal, D.D. Sharma Dairy animal production First edition 1995 International Book Distributing company, Lucknow

2 Sanjeev Sharma*, Aarti Bhardwaj$, Shalini Jain# and Hariom Yadav# *Animal Genetics and Breeding Division, #Animal Biochemistry Division, National Dairy Research Institute, Karnal-132001, Haryana, India $College of Applied Education and Health Sciences, Meerut, U.P. Source: Internet PowerPoint presentation

3 Venkatesh M.N. Research Journal Of BioTechnology Vol. 3 (1) Feb. (2008)

Res. J. Biotech.

4. http://www.ncbi.nlm.nih.gov/pubmed/15268796

Further readings

1. Singh BD (2007), Biotechnology Third edition, Kalyani Publishers, New Delhi

2. Kumar HD (2003), Modern concepts of Biotechnology, Vikas publishing house pvt ltd.